Probing the Influence of the Protein Scaffold on H-Cluster Reactivity via Gain-of-Function Studies─Improved H Evolution and O Tolerance through Rational Design of [FeFe] Hydrogenase.

[FeFe] hydrogenases make up a structurally diverse family of metalloenzymes that catalyze proton/dihydrogen interconversion. They can be classified into phylogenetically distinct groups denoted A-G, which differ in structure and reactivity. Prototypical Group A hydrogenases have high turnover rates and remarkable energy efficiency. As compared to Group A enzymes, the putatively sensory Group D hydrogenase from (HydS) has a thousand-fold lower H evolution rate and a high overpotential requirement to drive catalysis (irreversible) but shows increased inhibitor tolerance. This divergence in structure and activity between hydrogenases makes them ideal models for studying second (active-site environment) and outer (, substrate transport) coordination sphere effects on metal cofactors. Herein, we generated three HydS-based variants, each mimicking proposed key structural features of Group A hydrogenase: the "active site" (), "proton-transfer pathway" (), and "combined" ( = + ) variant. A fourth single-point variant, , which introduces a proposed critical cysteine in the active site, was characterized as a reference. No change in isolation resulted in Group A-like behavior; ., no positive impact on catalytic performance was observed. The variant, however, showed increased H evolution activity but retained the overpotential requirement. Additionally, the variant improved the already relatively high stability of HydS against O and CO inhibition. These findings show that activity rates, (ir)reversibility, and susceptibility to gaseous inhibitors are decoupled. Moreover, the results highlight the importance of exploring hydrogenase diversity as a path toward understanding the structural factors that enable the outstanding catalytic properties of [FeFe] hydrogenases.